Project Summary A central theme of research in the Cheng group is focused on basic understanding of how biomolecules and/or molecular assemblies function in space and time to drive life. For example, how does the membrane respond to the action potential in neurons? How is the metabolism remodelled during cell development or cancer progression? What happens to the chemistry inside a microorganism when exposed to a drug treatment? Answering these questions has broad implications for diagnosing and treating conditions ranging from infectious diseases to metastatic cancers. Towards this mission, Cheng and his team invent and apply highly sensitive chemical imaging technologies that are able to unveil hidden signatures in various living systems. The eventual goal is to enable molecule-based precision diagnosis and/or treatment of human diseases. The Cheng team further harnesses and manipulates the unique properties of photons to modulate the behaviour of cells. Two focused projects are photolysis of chromophores to eradicate drug-resistant bacteria and optoacoustic modulation of neural tissues at ultrahigh spatial precision. Overall, with integrated expertise in engineering, physics, chemistry, biology, medicine and entrepreneurship, the research team is devoted to three integrated thrusts: (1) Inventing label-free optical modulation and spectroscopic imaging technologies and pushing their physical limits; (2) Discovering molecular signatures that define cellular state and functions; (3) Converting label- free technologies and biological discoveries into molecule-based precision diagnosis and treatments. During the past 5 years (2013 to 2018), research by Cheng and co-workers has pushed the boundary of vibrational spectroscopic imaging in terms of speed, spectral bandwidth, imaging depth, and detection sensitivity (for a review, Science, 2015, 350: aaa8870). In parallel, via collaborations, Cheng and co-workers discovered significant metabolic signatures defining cancer aggressiveness (Cell Metabolism 2014), cancer cell stemness (Cell Stem Cell 2017), and antimicrobial resistance (Anal Chem 2017), as well as a spectroscopic indicator of membrane voltage in neurons (JPC Lett 2017). The overarching goal of this MIRA proposal is to further push the boundary of nonlinear vibrational spectroscopic imaging platforms in order to unveil the signatures that underlie initiation or progression of human diseases by ?watching the orchestra of molecules? in real space and time inside a living system. Cheng and co-workers will pursue this goal by advancing the capability of two complimentary vibrational imaging platforms, namely multiplex stimulated Raman scattering microscopy and infrared photothermal microscopy both invented in the Cheng lab, to reach single-molecule detection sensitivity, 100-nm spatial resolution, volumetric mapping at high speed, and deep-tissue penetration. As a focused application, the team will deploy the developed technologies to advance the basic understanding of how a biological cell reprograms its metabolism during development in vivo or in response to a stress.